Sentry guard cartridge

The native β-Lg concentration after heat treatment was determined by HPLC using the supernatant recovered after centrifugation (9000 rpm for 30 min at 4 °C) of the samples adjusted to pH 4.6. The chromatographic system was an Alliance e2695 Separation Module (Waters, Milford, MA, USA), composed by a XBridge Protein BEH C4 300 Å, 3.5 µm, 4.6 mm × 250 mm separation column associated with a guard column (Sentry Guard Cartridge, Waters). The two mobile phases used were 0.1% (v/v) of trifluoroacetic acid (99%, Acros Organics) in Milli-Q water as solvent A and 0.1% (v/v) trifluoroacetic acid, 80% acetonitrile (HPLC grade, Thermo Fisher Scientific) and 20% Milli-Q as solvent B. 20 µL of each sample was injected in the column maintained at 40 °C. The separation was performed at a flow rate of 1 mL/min with gradient elution and a detection wavelength of 215 nm. Analyses were repeated two times for each sample and three times for each standard. Calibration standards in the range from 0.25 to 3 g/L were prepared by dissolving a β-lg powder (BioPure industrial powder, Davisco, Foods International, Inc., Minnesota: β-lg at 88.85% purity) in Milli-Q water.
The native β-lg concentrations were calculated by averaging the measured chromatographic areas and converting each area into concentration by means of the calibration curve.

The chromatographic system consisted of an Alliance W2690 separation module equipped with an online degasser, an automatic injector, and a W2487 photodiode array detector set at 420 nm for the detection of pristimerin and tingenone. Data were collected and processed using Empower v.2 software for HPLC system (Waters, Milford, MA, USA). Separation was performed with a column Supelco Ascentis RP C18 (150 mm × 4.6 mm; particle size 5 µm, Sigma-Aldrich, St. Louis, MO, USA) column equipped with a Sentry Guard Cartridge (3.9 mm x 20 mm; Waters, Sigma-Aldrich, St. Louis, MO, USA) guard column. Both columns were maintained at 25 ± 1 °C. The mobile phase consisted of water with 0.4% of formic acid (v/v) (solvent A) and methanol (solvent B), using a gradient elution program for 10 min with a flow rate of 1.2 mL/min as follows: linear gradient ratio A/B 90:10 from the beginning of the chromatographic run to 6.0 min, A/B 90:10 to A/B 70:30 gradient from 6.01 min to 10.0 min, and finally, linear gradient ratio A/B 90:10 from 10.01 min to 15.0 min.